Investigating Novel Inhibitors of Epithelial - Mesenchymal Transition (EMT) and Cancer Stem Cells (CSCs) in Pancreatic Cancer

Pancreatic cancer is the 4th leading cause of cancer-related death in the U.S. in 2019. The five-year survival rate for all stages combined is only 9%, which is the lowest among all types of cancer. The current chemotherapeutic regimens for pancreatic cancer, including gemcitabine, FOLFIRNOX, and nab-paclitaxel, only provide limited benefits to the patients, due to low response rate and/or intolerable toxicities. Pancreatic cancer is enriched with cancer stem cells (CSCs), which contribute to its high metastatic tendency and resistance to treatments. The formation of CSCs is highly associated with cancer cell epithelial-mesenchymal transition (EMT), which contributes to the plasticity of cancer cells and comprises an initial step for metastasis. Inhibiting EMT and CSCs in pancreatic cancer could potentially inhibit pancreatic cancer progression, metastasis, and drug resistance. Various efforts have been made to develop anti-tumor agents through modulation of important signaling pathways involved in EMT and CSCs. However, there has not been a clinical success in a specific inhibitor to CSCs, or the EMT process. Extracts from medicinal plants have been rich sources for compounds possessing activities to attenuate CSC characteristics. These compounds contribute to the suppression of tumor progression and aggressiveness in various degrees. A great amount of synthetized small molecules have also been studied in targeting regulating factors of EMT and CSCs. Recent studies revealed that RNA binding proteins have the potential to serve as a therapeutic target in cancer management. The role of RNA binding proteins in EMT and CSCs is a research topic of increasing interests. The overall goal of this dissertation is to develop several approaches to discover and investigate novel EMT and CSC inhibitors for pancreatic cancer. We first investigated extracts of two traditional medicinal plants, Pao Pereira extract (Pao) and Rauwolfia vomitoria extract (Rau) for their anti-pancreatic CSC activities. Data demonstrated that Pao and Rau preferentially inhibited the viability of pancreatic CSC population, compared with the whole cancer cell population. Consistently, the expression of CSC-related genes, DPPA4, ESRRB and TCL1A, and β-catenin target genes, BCL2L2 and COX2 were significantly suppressed with Pao and Rau treatment. The tumorigenicity was also inhibited by Pao and Rau in subcutaneous pancreatic cancer xenograft mouse models. Pao and Rau demonstrated potent anti-pancreatic CSC effect in vitro and in vivo and are worth further investigation. (Chapter 4 and Chapter 5) Next, we investigated the role of an RNA binding protein, HuR, in pancreatic cancer EMT and CSCs. Our data showed that knockdown of HuR in pancreatic cancer cells significantly inhibited cell migration, invasion, and stem-like features. Consistently, knockdown of HuR led to the inhibition of EMT by upregulating epithelium marker Claudin-1 and downregulating mesenchymal marker Vimentin and Snail. Restoration of HuR in HuR deleted (KO) pancreatic cancer cells enhanced migration and enriched CSC population. RNP-IP assay and dual-luciferase reporter assay identified that SNAI1 mRNA directly bound to HuR through an AU-rich elements (AREs) dependent way. The data demonstrated that inhibition of HuR destabilized SNAI1 mRNA, and subsequently reduced Snail protein levels, resulting in the EMT and CSCs inhibition in pancreatic cancer cells. (Chapter 6) We then developed a small molecule inhibitor of HuR, KH-3, and evaluated the activities of KH-3 on pancreatic cancer EMT and CSCs. Our data showed that KH-3 directly bound to HuR and interfered the binding of HuR with its target mRNAs. As an HuR/mRNA disruptor, KH-3 treatment successfully mimicked the effects of HuR knockdown in pancreatic cancer cell lines: KH-3 significantly inhibited pancreatic cancer cell migration, invasion, tumor sphere formation and EMT. In an orthotopic pancreatic cancer mouse model, KH-3 significantly inhibited tumor growth and metastasis. Importantly, loss of target analyses showed that KH-3 exerted its anti-pancreatic cancer effect in an HuR dependent way in vitro and in vivo. In conclusion, KH-3 serves as a potent and specific HuR/mRNA disruptor and inhibits pancreatic cancer EMT and CSCs in vitro and in vivo. As the first of its class, KH-3 can serve as a drug-lead for developing agents that comprehensively inhibit pancreatic cancer growth, metastasis, and treatment resistance. (Chapter 7) Overall, studies in this dissertation resulted in the identification of three novel EMT and CSC inhibitors, which can be further investigated in pre-clinical and clinical studies for their anti-tumor efficacy. These three EMT and CSC inhibitors each have their own merits and shortcomings. Natural plant extracts, Pao and Rau, have potent anti-pancreatic CSC activities with limited toxicity, thus hold the potential to combine with conventional chemotherapy agents. However, as compounds mixtures, Pao and Rau need future purification and isolation for clinical applications. The compound KH-3 was identified as a novel HuR inhibitor and provided a novel drug-lead for pancreatic cancer management. More studies on the pharmacokinetics, pharmacodynamics, and the combination of KH-3 with conventional chemotherapeutic agents are worth investigation. We hope that the work we have accomplished in this dissertation could add to our knowledge of a new mechanism on EMT and CSC regulation and contribute towards discovery of new drugs for pancreatic cancer